低磷胁迫大豆SSH文库构建与分析
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摘要
大豆是重要粮食油料作物,在国民经济中占有重要地位;大豆需磷量较大,我国大多数土壤有效磷含量较低,限制了大豆产量;利用转基因技术培育磷高效品种是改善磷营养状况的有效途径,但目前可利用的磷高效基因数量有限。针对这一问题,本研究以耐低磷品种“中黄15”为材料,利用SSH技术,构建低磷诱导大豆根系cDNA文库,为进一步挖掘磷高效基因创建平台,为解析磷高效机制奠定基础。主要研究结果如下:
1.成功构建了大豆耐低磷品种“中黄15”低磷胁迫SSH文库。文库滴度为4.3×105 cfu.mL-1,阳性克隆比为75%,符合一般文库要求,且质量较高。该文库保存阳性克隆1500个,随机挑取500个阳性克隆分析发现,文库插入片段大小在200-1000 bp之间,平均长度在500 bp左右。
2.完成了文库中200个阳性克隆的测序工作。获得非重复ESTs序列150个,对其进行同源性比对,BLASTx分析发现有79条ESTs序列与已知功能蛋白序列同源性较高,占所有测序ESTs的52.3%;经BLASTn分析,有70条ESTs序列与已知功能的ESTs序列具有较高同源性,占所有测序ESTs的47.0%。
3.详细分析了所获得的低磷胁迫表达ESTs序列。通过对测序结果进行比对发现,所比对的已知功能同源基因涉及了植物细胞内的多种代谢和应答过程,其中包括参与细胞内的新陈代谢、信号传导、抗病防御、蛋白质合成、蛋白质加工以及细胞生长/分裂等代谢和应答过程。在所有的代谢和应答基因中,以细胞内的抗病/防御类基因所占比例最大,其比例为29.1%;其次以细胞内的信号传导途径基因较多,占所分析基因的13.9%。
4.综合分析了与植物在低磷条件下的耐低磷特性相关的ESTs序列。主要包括:与菜豆液泡ATPase亚基E同源EST序列(180号)、与蒺藜苜蓿乙醛脱氢酶同源EST序列(61号)、促分裂原活化蛋白质激酶(114号)、酪氨酸蛋白激酶(I24号)、与硫氧还蛋白相关异构体Ⅰ同源EST序列(F5号)、与大豆bZIP转录因子同源EST序列(113号)、与豇豆EIL2转录因子同源EST序列(K3号)、过氧化物酶(38号)、阳离子过氧化物酶Ⅱ(K1)、过氧化物酶甜菜碱醛脱氢酶(134号)等。
Soybean is an important food and economic crop, and plays a very important role in the national economy. It is also a kind of crops which need a large amount of phosphorus, and its production is significantly limited by low phosphorus availability in the soil. Using the transgenic technology to breed high-efficient phosphorus variety is an effective method to improve the nutritional status of phosphorus in soybean. But the limited quantity of related genes is a major bottleneck. To mine and clone more phosphorus-metabolism related genes, in present study, a high quality soybean root cDNA library of low phosphorus-tolerance variety Zhong-Huang 15 was constructed under the low phosphorus stress by using suppression subtractive hybridization (SSH) technique. The results were summarized as follows:
1. The results showed that the titer of the SSH cDNA library was 4.3×105 cfu.mL-1, and transformation efficiency was 75%. There were 1500 positive clones stored in this library, and the size of insert fragments was ranged from 200 bp to 1000 bp with a average size 500 bp, which was determined by positive clones randomly.
2. Two hundred positive clones selected randomly were sequenced in the constructed SSH library and 150 unique ESTs were obtained. Then the ESTs similarity was analyzed via BLASTx and BLASTn software and there were 79 ESTs of homology with the known function protein sequences. The results of BLASTn confirmed that there were 70 ESTs homologous with the known ESTs sequences.
3. Analysis results of the obtained soybean genes in the library induced by the low phosphorus stress showed that the known functions of the homologous genes were involved in many metabolism and response processes such as metabolism, signal transduction, disease/defence, protein synthesis, processing, and cell growth / division etc. The results also showed that the disease / defense category gene products shared a maximum of 29.1%, which was the largest type, and the signal transduction pathway genes fell into another large type, which accounted for13.9% of the total ESTs.
4. Analyzed the Blast results of the sequenced ESTs, we found that some ESTs were homologous to the related genes of plant tolerance to low phosphorus stress. These homologous ESTs were as follows: EST 180 ( homologous to the vacuolar ATPase subunit E in Phaseolus acutifolius), EST 61 ( homologous to the Aldehyde dehydrogenase in Medicago truncatula), EST 114 ( homologous to MAPK in Glycine max), EST I24 ( homologous to Tyrosine kinase in Glycine max), EST F5 ( homologous to Similar to thioredoxin-related isoformⅠin Vitis vinifera), EST 113 ( homologous to bZIP transcription factor bZIP41 in Glycine max), EST K3 ( homologous to transcription factor EIL2 in Vigna radiata), EST 38 ( homologous to peroxidase in Dimocarpus longan), EST K1 ( homologous to Cationic peroxidaseⅡin Arachis hypogaea) and EST 134 ( homologous to peroxisomal betaine-aldehyde dehydrogenase in Glycine max).
1.成功构建了大豆耐低磷品种“中黄15”低磷胁迫SSH文库。文库滴度为4.3×105 cfu.mL-1,阳性克隆比为75%,符合一般文库要求,且质量较高。该文库保存阳性克隆1500个,随机挑取500个阳性克隆分析发现,文库插入片段大小在200-1000 bp之间,平均长度在500 bp左右。
2.完成了文库中200个阳性克隆的测序工作。获得非重复ESTs序列150个,对其进行同源性比对,BLASTx分析发现有79条ESTs序列与已知功能蛋白序列同源性较高,占所有测序ESTs的52.3%;经BLASTn分析,有70条ESTs序列与已知功能的ESTs序列具有较高同源性,占所有测序ESTs的47.0%。
3.详细分析了所获得的低磷胁迫表达ESTs序列。通过对测序结果进行比对发现,所比对的已知功能同源基因涉及了植物细胞内的多种代谢和应答过程,其中包括参与细胞内的新陈代谢、信号传导、抗病防御、蛋白质合成、蛋白质加工以及细胞生长/分裂等代谢和应答过程。在所有的代谢和应答基因中,以细胞内的抗病/防御类基因所占比例最大,其比例为29.1%;其次以细胞内的信号传导途径基因较多,占所分析基因的13.9%。
4.综合分析了与植物在低磷条件下的耐低磷特性相关的ESTs序列。主要包括:与菜豆液泡ATPase亚基E同源EST序列(180号)、与蒺藜苜蓿乙醛脱氢酶同源EST序列(61号)、促分裂原活化蛋白质激酶(114号)、酪氨酸蛋白激酶(I24号)、与硫氧还蛋白相关异构体Ⅰ同源EST序列(F5号)、与大豆bZIP转录因子同源EST序列(113号)、与豇豆EIL2转录因子同源EST序列(K3号)、过氧化物酶(38号)、阳离子过氧化物酶Ⅱ(K1)、过氧化物酶甜菜碱醛脱氢酶(134号)等。
Soybean is an important food and economic crop, and plays a very important role in the national economy. It is also a kind of crops which need a large amount of phosphorus, and its production is significantly limited by low phosphorus availability in the soil. Using the transgenic technology to breed high-efficient phosphorus variety is an effective method to improve the nutritional status of phosphorus in soybean. But the limited quantity of related genes is a major bottleneck. To mine and clone more phosphorus-metabolism related genes, in present study, a high quality soybean root cDNA library of low phosphorus-tolerance variety Zhong-Huang 15 was constructed under the low phosphorus stress by using suppression subtractive hybridization (SSH) technique. The results were summarized as follows:
1. The results showed that the titer of the SSH cDNA library was 4.3×105 cfu.mL-1, and transformation efficiency was 75%. There were 1500 positive clones stored in this library, and the size of insert fragments was ranged from 200 bp to 1000 bp with a average size 500 bp, which was determined by positive clones randomly.
2. Two hundred positive clones selected randomly were sequenced in the constructed SSH library and 150 unique ESTs were obtained. Then the ESTs similarity was analyzed via BLASTx and BLASTn software and there were 79 ESTs of homology with the known function protein sequences. The results of BLASTn confirmed that there were 70 ESTs homologous with the known ESTs sequences.
3. Analysis results of the obtained soybean genes in the library induced by the low phosphorus stress showed that the known functions of the homologous genes were involved in many metabolism and response processes such as metabolism, signal transduction, disease/defence, protein synthesis, processing, and cell growth / division etc. The results also showed that the disease / defense category gene products shared a maximum of 29.1%, which was the largest type, and the signal transduction pathway genes fell into another large type, which accounted for13.9% of the total ESTs.
4. Analyzed the Blast results of the sequenced ESTs, we found that some ESTs were homologous to the related genes of plant tolerance to low phosphorus stress. These homologous ESTs were as follows: EST 180 ( homologous to the vacuolar ATPase subunit E in Phaseolus acutifolius), EST 61 ( homologous to the Aldehyde dehydrogenase in Medicago truncatula), EST 114 ( homologous to MAPK in Glycine max), EST I24 ( homologous to Tyrosine kinase in Glycine max), EST F5 ( homologous to Similar to thioredoxin-related isoformⅠin Vitis vinifera), EST 113 ( homologous to bZIP transcription factor bZIP41 in Glycine max), EST K3 ( homologous to transcription factor EIL2 in Vigna radiata), EST 38 ( homologous to peroxidase in Dimocarpus longan), EST K1 ( homologous to Cationic peroxidaseⅡin Arachis hypogaea) and EST 134 ( homologous to peroxisomal betaine-aldehyde dehydrogenase in Glycine max).
引文
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